Despite concerted efforts to control virus-associated diseases worldwide, they still remain a major public health problem. The host's immediate response to viral infections relies on pattern recognition receptors (PRRs) that sense nucleic acids or other conserved structural components of invading viruses and subsequently activate signaling cascades leading to interferon (IFN)- mediated antiviral defense mechanisms. Melanoma differentiation-associated gene 5 (MDA5) has emerged as a key cytosolic receptor for sensing RNA viruses, including picornaviruses and certain Flaviviruses. Upon binding of viral RNA, the N-terminal caspase recruitment domains (CARDs) of MDA5 interact with MAVS/VISA/IPS-1/Cardif downstream partner to trigger type I IFN production to prevent completion of the virus lifecycle as well as virus dissemination in vivo. Despite the recent rapid progress in deciphering molecular components in the MDA5 signaling pathway, the regulation of its antiviral activity remains largely undetermined. However, it is exactly the regulation of the host IFN production, which dictates the outcome of the viral infection as well as severity of disease and pathogenesis. Thus, the proposed study is directed toward investigating how the signal transducing activity of the cytosolic viral RNA receptor MDA5 is regulated by host-encoded factors to modulate IFN-mediated host responses. Biochemical, cell biological and structural studies will focus on defining in mechanistic detail how posttranslational modifications affect the MDA5 signal transducing activity to limit viral replication (Aim 1). This study will further aim at identifying novel host cell factors for regulating MDA5 antiviral function, and at investigating their roles in the MDA5-mediated immune surveillance against RNA virus infections (Aim 2). Insights gained from this study will not only greatly expand our understanding of the regulatory networks of the innate immune system, but also identify novel signaling molecules that play key roles in antiviral innate immunity, thereby providing potential targets for therapeutic intervention against viral infections. PUBLIC HEALTH RELEVANCE: Despite concerted efforts to control virus-associated diseases worldwide, they still remain a major public health problem. The cytosolic viral RNA sensor MDA5 plays a pivotal role in host cell defense against viral pathogens. The proposed study is targeted to delineate the molecular mechanisms underlying virus recognition and host innate immunity to viral infections, with a specific focus on the MDA5-mediated interferon response.